Inherited epidermolysis bullosa (EB) is a mechanobullous disease, characterized by the development of the blisters following minor or insignificant trauma of the skin. Based on the tissue separation level, it can be divided into three major forms: simplex, junctional and dystrophic. In junctional EB (JEB), skin separation occurs within the lamina lucida of the cutaneous basement membrane zone (BMZ) and is associated with weak adhesion of basal keratinocytes to dermal anchoring fibrils. It is often caused by dysfunction of a connective tissue protein laminin 332, which in turn, results from mutations in LAMA3, LAMB3 or LAMC2 genes encoding 13, 23, and 32 subunits of this protein, respectively. At present, gene therapy approaches are not readily available for effective treatment of JEB. For the JEB patients, day-to-day management of the disease is revolving around prevention of mechanical trauma and infection via the use of protective loose garments and antibiotic creams and ointments. Previously we proposed and recently demonstrated that individual chains of laminin 332 (23 in particular) can successfully replace the absent chains to restore intracellular assembly of the trimeric laminin in keratinocytes isolated from patients suffering from Herlitz type JEB (H-JEB) in vitro. Also, using organotypic cultures of the H-JEB human skin, we showed that hybrid laminin 332 that contains endogenous 13, 32 and recombinant 23 subunits can restore the adhesion of the basal keratinocytes to the dermal anchoring fibrils. Therefore, based on these data, here we hypothesized that recombinant trimeric laminin 332 can be used as a therapeutic for the restoration of the dermal-epidermal junction and for a localized treatment of any form of the laminin 332-dependent JEB. To experimentally test the proposed hypothesis we will: (1) Optimize large-scale in vitro production and purification of the biologically active, recombinant laminin 332 of human and mouse origins;(2)Test the ability of the recombinant protein to interact with 1624 integrins and type VII collagen and restore the adhesion of the protein-treated keratinocytes in vitro and in organotypic cultures of human JEB skin, and (3) Characterize pharmacokinetics of the laminin 332-based therapeutics on existing JEB pre-clinical mouse models. Overall, the completion of the proposed projects will evaluate the feasibility of the proposed therapeutic approach and if successful, will allow us to further develop the proposed strategy toward its direct clinical application. Inherited epidermolysis bullosa is a mechanobullous disease, characterized by the development of the blisters following minor or insignificant trauma of the skin which is often associated with the dysfunction of the connective tissue protein, laminin 332. In the current project we proposed to utilize the recombinant laminin 332 for the localized treatment of this devastating disorder. If the proposed therapy will be positively evaluated during these studies, it may become a powerful and relatively inexpensive therapeutic approach for treatment of this devastating disorder and benefit public health.